Investigating the role of N-WASP in the development of colorectal cancer

Colorectal cancer (CRC) is the second most common cancer in Europe, with the second highest mortality rate. Although prognosis is improving, survival rates remain poor for those presenting with the most advanced stages of the disease. There is therefore a need for improved early diagnosis and thus a greater understanding of the early stages of the development of colorectal tumours is desirable. Additionally, as most deaths in colorectal cancer are due to advanced metastatic disease, it is of great interest to explore any potential mechanisms by which metastatic disease can be inhibited. N-WASP is a ubiquitously expressed protein with multiple intracellular roles including actin regulation and maintaining stability of epithelial cell-cell junctions. Through its role as an actin regulator, it has been implicated in the processes of invasion and metastasis of multiple cancer types. Its role in the development and progression of colorectal cancer however has not been fully explored. This thesis will present a series of in vitro and in vivo studies that were carried out with the aim of answering the following questions: • Does N-Wasp have a role in normal intestinal homeostasis? • Does N-Wasp knockout affect the development of tumours in a mouse model of intestinal tumourigenesis? • Does N-Wasp knockout affect the invasive properties of intestinal cancer in vitro? • Does N-WASP correlate with prognosis or other indicators in human colorectal cancer TMAs? Findings from the in vivo experiments, using an inducible, gut-specific knockout model, have uncovered potential roles for N-Wasp in regulating differentiation and migration of intestinal epithelial cells. Although it had no effect in short term models of intestinal hyperproliferation, N-Wasp knockout increased tumour burden and decreased survival in an established in vivo model of intestinal tumourigenesis, in which there is heterozygous loss of Apc (Apcfl/+). No effect was seen on tumour development or survival when additional N-WASP knockout was introduced into a more rapid model, with heterozygous loss of Apc and mutation of Kras (Apcfl/+ KrasG12D/+). N-WASP expression in human colorectal cancer was assessed using immunohistochemical staining of two tissue microarrays. Low levels of N-WASP expression were found to be associated with presence of MMR deficiency. There was no statistically significant difference in overall or cancer specific survival based on N-WASP expression. Collectively, the data presented here suggest a previously unreported role for N-WASP in regulation of intestinal epithelial differentiation and indicate that it may act as a tumour suppressor against development of benign precursor lesions of colorectal cancer. Further research is warranted to delineate the mechanisms underlying these processes.

Theta-associated high-frequency oscillations (110–160 Hz) in the hippocampus and neocortex

TORT, A. B. L. ; SCHEFFER-TEIXEIRA, R ; Souza, B.C. ; DRAGUHN, A. ; BRANKACK, J. . Theta-associated high-frequency oscillations (110-160 Hz) in the hippocampus and neocortex. Progress in Neurobiology , v. 100, p. 1-14, 2013.

Superconducting Transition-Edge Sensor Physics

Despite record-setting performance demonstrated by superconducting Transition Edge Sensors (TESs) and growing utilization of the technology, a theoretical model of the physics governing TES devices superconducting phase transition has proven elusive. Earlier attempts to describe TESs assumed them to be uniform superconductors. Sadleir et al. 2010 shows that TESs are weak links and that the superconducting order parameter strength has significant spatial variation. Measurements are presented of the temperature T and magnetic field B dependence of the critical current Ic measured over 7 orders of magnitude on square Mo/Au bilayers ranging in length from 8 to 290 microns. We find our measurements have a natural explanation in terms of a spatially varying order parameter that is enhanced in proximity to the higher transition temperature superconducting leads (the longitudinal proximity effect) and suppressed in proximity to the added normal metal structures (the lateral inverse proximity effect). These in-plane proximity effects and scaling relations are observed over unprecedentedly long lengths (in excess of 1000 times the mean free path) and explained in terms of a Ginzburg-Landau model. Our low temperature Ic(B) measurements are found to agree with a general derivation of a superconducting strip with an edge or geometric barrier to vortex entry and we also derive two conditions that lead to Ic rectification. At high temperatures the Ic(B) exhibits distinct Josephson effect behavior over long length scales and following functional dependences not previously reported. We also investigate how film stress changes the transition, explain some transition features in terms of a nonequilibrium superconductivity effect, and show that our measurements of the resistive transition are not consistent with a percolating resistor network model.

Un micro-bolomètre supraconducteur: de la conception à l'utilisation au laboratoire

Pour augmenter sa compréhension du monde qui l’entoure, le physicien moderne ne cesse de travailler au développement d’outils théoriques ou expérimentaux pour l’aider à répondre à ses questions fondamentales. Une partie de ces chercheurs tente de répondre à une question bien définie, mais simultanément très vague : qu’est-ce que le bruit électro- nique? Guidés par cette idée, certains étudient des dispositifs comme la jonction tunnel ou la jonction Josephson, alors que d’autres travaillent à l’amélioration des méthodes de détection du bruit. Le présent mémoire de maîtrise traite donc de la conception d’un outil de détection bien particulier, le micro-bolomètre supraconducteur de niobium-titane-niobium. La théorie derrière le fonctionnement d’un tel dispositif est expliquée à l’aide d’une comparaison entre un bolomètre conventionnel et un bolomètre supraconducteur. Des concepts comme la sensibilité d’un détecteur, la conductance thermique et la méthode d’utilisation sont présentés. Les étapes du procédé de fabrication sont ensuite explicitées dans les moindres détails. Finalement, les propriétés électroniques d’un tel micro-bolomètre sont analysées à l’aide de la courbe caractéristique courant-tension, de la courbe de transition supraconductrice en température et de dfférentes mesures en réflectométrie. La puissance équivalente de bruit (NEP) mesurée est de l’ordre de 10[indice supérieur −17] W/√Hz et le temps caractéristique de détection est de 1.43 μs. Le dispositif présenté dans ce mémoire a un avantage important par rapport aux bolomètres supraconducteurs généralement utilisés : il ne nécessite pas de courant de polarisation continu pour le mettre en fonctionnement. Ceci peut résulter en divers avantages technologiques.

Zbadanie prądu tunelowego w złączach typu metal-nadprzewodnik trypletowy dla wybranych symetrii parametru porządku

Wydział Fizyki: Zakład Teorii Ciała Stałego

Characterization of in vitro effects of patulin on intestinal epithelial and immune cells

The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12 mM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50 mM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50 mM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24 h of exposure to 50 mM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10 nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins

Proliferative Defects and Formation of a Double Cortex in Mice Lacking Mltt4 and Cdh2 in the Dorsal Telencephalon

Radial glial cells (RGCs) in the ventricular neuroepithelium of the dorsal telencephalon are the progenitor cells for neocortical projection neurons and astrocytes. Here we showthatthe adherens junction proteins afadin and CDH2 are criticalforthe control of cell proliferation in the dorsal telencephalon and for the formation of its normal laminar structure. Inactivation of afadin or CDH2 in the dorsal telenceph-alon leads to a phenotype resembling subcortical band heterotopia, also known as “double cortex,” a brain malformation in which heterotopic gray matter is interposed between zones of white matter. Adherens junctions between RGCs are disrupted in the mutants, progenitor cells are widely dispersed throughout the developing neocortex, and their proliferation is dramatically increased. Major subtypes of neocortical projection neurons are generated, but their integration into cell layers is disrupted. Our findings suggest that defects in adherens junctions components in mice massively affects progenitor cell proliferation and leads to a double cortex-like phenotype.

NETCODE: an XOR-based warning dissemination scheme for vehicular wireless networks

The next generation of vehicles will be equipped with automated Accident Warning Systems (AWSs) capable of warning neighbouring vehicles about hazards that might lead to accidents. The key enabling technology for these systems is the Vehicular Ad-hoc Networks (VANET) but the dynamics of such networks make the crucial timely delivery of warning messages challenging. While most previously attempted implementations have used broadcast-based data dissemination schemes, these do not cope well as data traffic load or network density increases. This problem of sending warning messages in a timely manner is addressed by employing a network coding technique in this thesis. The proposed NETwork COded DissEmination (NETCODE) is a VANET-based AWS responsible for generating and sending warnings to the vehicles on the road. NETCODE offers an XOR-based data dissemination scheme that sends multiple warning in a single transmission and therefore, reduces the total number of transmissions required to send the same number of warnings that broadcast schemes send. Hence, it reduces contention and collisions in the network improving the delivery time of the warnings. The first part of this research (Chapters 3 and 4) asserts that in order to build a warning system, it is needful to ascertain the system requirements, information to be exchanged, and protocols best suited for communication between vehicles. Therefore, a study of these factors along with a review of existing proposals identifying their strength and weakness is carried out. Then an analysis of existing broadcast-based warning is conducted which concludes that although this is the most straightforward scheme, loading can result an effective collapse, resulting in unacceptably long transmission delays. The second part of this research (Chapter 5) proposes the NETCODE design, including the main contribution of this thesis, a pair of encoding and decoding algorithms that makes the use of an XOR-based technique to reduce transmission overheads and thus allows warnings to get delivered in time. The final part of this research (Chapters 6--8) evaluates the performance of the proposed scheme as to how it reduces the number of transmissions in the network in response to growing data traffic load and network density and investigates its capacity to detect potential accidents. The evaluations use a custom-built simulator to model real-world scenarios such as city areas, junctions, roundabouts, motorways and so on. The study shows that the reduction in the number of transmissions helps reduce competition in the network significantly and this allows vehicles to deliver warning messages more rapidly to their neighbours. It also examines the relative performance of NETCODE when handling both sudden event-driven and longer-term periodic messages in diverse scenarios under stress caused by increasing numbers of vehicles and transmissions per vehicle. This work confirms the thesis' primary contention that XOR-based network coding provides a potential solution on which a more efficient AWS data dissemination scheme can be built.

Simulation tools for the analysis of single electronic systems

Developments in theory and experiment have raised the prospect of an electronic technology based on the discrete nature of electron tunnelling through a potential barrier. This thesis deals with novel design and analysis tools developed to study such systems. Possible devices include those constructed from ultrasmall normal tunnelling junctions. These exhibit charging effects including the Coulomb blockade and correlated electron tunnelling. They allow transistor-like control of the transfer of single carriers, and present the prospect of digital systems operating at the information theoretic limit. As such, they are often referred to as single electronic devices. Single electronic devices exhibit self quantising logic and good structural tolerance. Their speed, immunity to thermal noise, and operating voltage all scale beneficially with junction capacitance. For ultrasmall junctions the possibility of room temperature operation at sub picosecond timescales seems feasible. However, they are sensitive to external charge; whether from trapping-detrapping events, externally gated potentials, or system cross-talk. Quantum effects such as charge macroscopic quantum tunnelling may degrade performance. Finally, any practical system will be complex and spatially extended (amplifying the above problems), and prone to fabrication imperfection. This summarises why new design and analysis tools are required. Simulation tools are developed, concentrating on the basic building blocks of single electronic systems; the tunnelling junction array and gated turnstile device. Three main points are considered: the best method of estimating capacitance values from physical system geometry; the mathematical model which should represent electron tunnelling based on this data; application of this model to the investigation of single electronic systems. (DXN004909)

Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient.

Stenosis triggers spread of helical Pseudomonas biofilms in cylindrical flow systems

Biofilms are multicellular bacterial structures that adhere to surfaces and often endow the bacterial population with tolerance to antibiotics and other environmental insults. Biofilms frequently colonize the tubing of medical devices through mechanisms that are poorly understood. Here we studied the helicoidal spread of Pseudomonas putida biofilms through cylindrical conduits of varied diameters in slow laminar flow regimes. Numerical simulations of such flows reveal vortical motion at stenoses and junctions, which enhances bacterial adhesion and fosters formation of filamentous structures. Formation of long, downstream-flowing bacterial threads that stem from narrowings and connections was detected experimentally, as predicted by our model. Accumulation of bacterial biomass makes the resulting filaments undergo a helical instability. These incipient helices then coarsened until constrained by the tubing walls, and spread along the whole tube length without obstructing the flow. A three-dimensional discrete filament model supports this coarsening mechanism and yields simulations of helix dynamics in accordance with our experimental observations. These findings describe an unanticipated mechanism for bacterial spreading in tubing networks which might be involved in some hospital-acquired infections and bacterial contamination of catheters.

Shortcuts to adiabaticity in the double well

123 p.

High On/Off ratio memristive switching of manganite/cuprate bilayer by interfacial magnetoelectricity

Memristive switching serves as the basis for a new generation of electronic devices. Memristors are two-terminal devices in which the current is turned on and off by redistributing point defects, e.g., vacancies, which is difficult to control. Memristors based on alternative mechanisms have been explored, but achieving both the high On/Off ratio and the low switching energy desirable for use in electronics remains a challenge. Here we report memristive switching in a La_(0.7)Ca_(0.3)MnO_(3)/PrBa_(2)Cu_(3)O_(7) bilayer with an On/Off ratio greater than 103 and demonstrate that the phenomenon originates from a new type of interfacial magnetoelectricity. Using results from firstprinciples calculations, we show that an external electric-field induces subtle displacements of the interfacial Mn ions, which switches on/off an interfacial magnetic “dead” layer, resulting in memristive behavior for spin-polarized electron transport across the bilayer. The interfacial nature of the switching entails low energy cost about of a tenth of atto Joule for write/erase a “bit”. Our results indicate new opportunities for manganite/cuprate systems and other transition-metal-oxide junctions in memristive applications.

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Clostridium difficile-associated disease causes diarrhea to fulminant colitis and death. We investigated the role of phospholipase A(2) (PLA(2)) inhibitors, aristolochic acid (AA), bromophenacyl bromide BPB and quinacrine (QUIN) on the C. difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion. Toxin A caused severe hemorrhagic and inflammatory fluid secretion at 6-8 h in rabbit ileal segments, an effect that was significantly inhibited by QUIN (71%, P < 0.01), AA (87%, P < 0.0001) or by BPB (51%, P < 0.01). The secretory effect of toxin A was also inhibited in segments adjacent to those with AA (89%, P < 0.01). Furthermore, QUIN or AA substantially reduced the histologic damage seen after 6-8 h in rabbit ileal segments. The cyclooxygenase inhibitor, indomethacin, also significantly inhibited (96%; n = 6) the secretory effects of toxin A in ligated rabbit intestinal segments. The destruction by toxin A of F-actin at the light junctions of T-84 cell monolayers was not inhibited by AA or BPB. AA or QUIN had no effect on the T-84 cell tissue resistance reduction over 8-24 h after toxin A exposure. All the inhibitors were shown to be effective in the doses administered direct in ileal loops to inhibit PLA(2) activity. The data suggest that PLA(2) is involved in the major pathway of toxin A-induced histologic inflammatory damage and hemorrhagic fluid secretion. Cop. right (C) 2008 John Wiley & Sons, Ltd.

Electronic levels in MEH-PPV

pn-Junctions of MEH-PPV on top of heavily doped n-type silicon were used in electrical measurements. Through deep-level transient-spectroscopy (DLTS)-like measurements, four traps (two majority and two minority traps) could be identified on top of the shallow acceptor level responsible for conduction. Furthermore, evidence is found for interface states. (C) 2000 Published by Elsevier Science S.A. All rights reserved.